Marfan syndrome is an autosomal dominant systemic disorder of the connective tissue. Children affected by the Marfan syndrome carry a mutation in one of their two copies of the gene that encodes the connective tissue protein fibrillin-1. Marfan syndrome affects most organs and tissues, especially the skeleton, lungs, eyes, heart, and the large blood vessel that distributes blood from the heart to the rest of the body. A case report of Marfan syndrome has been reported with oral features. The dental problems of the child were treated under general anesthesia and a one-month review showed intact stainless steel crowns' restorations and no signs of secondary caries.
Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RACF28L) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 μs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue.
Generally, limited research is extended in studying stability and applicational properties of silver nanoparticles (Ag NPs) synthesized by adopting ‘green chemistry’ protocol. In this work, we report on the synthesis of stable Ag NPs using plant-derived materials such as leaf extract of Neem (Azadirachta indica) and biopolymer pectin from apple peel. In addition, the applicational properties of Ag NPs such as surface-enhanced Raman scattering (SERS) and antibacterial efficiencies were also investigated. As-synthesized nanoparticles (NPs) were characterized using various instrumentation techniques. Both the plant materials (leaf extract and biopolymer) favored the synthesis of well-defined NPs capped with biomaterials. The NPs were spherical in shape with an average particle size between 14-27 nm. These bio-NPs exhibited colloidal stability in most of the suspended solutions such as water, electrolyte solutions (NaCl; NaNO3), biological solution (bovine serum albumin), and in different pH solutions (pH 7; 9) for a reasonable time period of 120 hrs. Both the bio-NPs were observed to be SERS active through displaying intrinsic SERS signals of the Raman probe molecule (Nile blue A). The NPs were effective against the Escherichia coli bacterium when tested in nutrient broth and agar medium. Scanning and high-resolution transmission electron microscopy (SEM and HRTEM) images confirmed cellular membrane damage of nanoparticle treated E. coli cells. These environmental friendly template Ag NPs can be used as an antimicrobial agent and also for SERS based analytical applications.
Chemotherapy is commonly used in cancer treatments, however only 25% of cancers are responsive and a significant proportion develops resistance. The p53 tumour suppressor is crucial for cancer development and therapy, but has been less amenable to therapeutic applications due to the complexity of its action, reflected in 66,000 papers describing its function. Here we provide a systematic approach to integrate this information by constructing a large-scale logical model of the p53 interactome using extensive database and literature integration. The model contains 206 nodes representing genes or proteins, DNA damage input, apoptosis and cellular senescence outputs, connected by 738 logical interactions. Predictions from in silico knock-outs and steady state model analysis were validated using literature searches and in vitro based experiments. We identify an upregulation of Chk1, ATM and ATR pathways in p53 negative cells and 61 other predictions obtained by knockout tests mimicking mutations. The comparison of model simulations with microarray data demonstrated a significant rate of successful predictions ranging between 52% and 71% depending on the cancer type. Growth factors and receptors FGF2, IGF1R, PDGFRB and TGFA were identified as factors contributing selectively to the control of U2OS osteosarcoma and HCT116 colon cancer cell growth. In summary, we provide the proof of principle that this versatile and predictive model has vast potential for use in cancer treatment by identifying pathways in individual patients that contribute to tumour growth, defining a sub population of “high” responders and identification of shifts in pathways leading to chemotherapy resistance.
Among the viruses infecting penaeid shrimp, monodon-type baculovirus (MBV) otherwise known as Penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV), is one of the widely reported and well described viruses. It is a rod-shaped, enveloped, double-stranded DNA virus, and considered till recently, as the type A baculovirus. Besides MBV, two strains of SNPV are reported—plebejus baculovirus and bennettae baculovirus. MBV was reported to be originated from Taiwan and has wide geographic distribution and is reported to be enzootic in wild penaeids of the Indo-pacific coasts of Asia. The virus also has diverse host-range including a variety of cultured and captured shrimp species and freshwater prawn, Macrobrachium rosenbergii. MBV has been reported in all life stages of P. monodon with late larval, postlarval and young juvenile as the most susceptible stages/ages. However, MBV has not been documented in early larval stages. Although MBV has been reported to be tolerated well by shrimp, the infection has been attributed to decreased productivity. The target organs or tissues of MBV are the hepatopancreatic tubules and duct epithelium of postlarvae, juveniles and adults, and the anterior midgut epithelium of very young postlarvae. The prominent clinical sign of infection is the presence of multiple spherical inclusion bodies in the hepatopancreas and midgut epithelial cells. The major mode of transmission of the virus is horizontal through oral exposure to occlusion bodies, contaminated tissues or fomites. Minor morphometric variation of the virus has been reported among different isolates. The rod-shaped enveloped virus particles range from 265–324 nm in length and 42–77 nm in diameter. Although complete genome sequence of MBV is not available, nucleic acid of MBV is circular, double-stranded DNA with a genome size ranging from 80 to 160 kbp. The virus codes for a 53 kDa major polyhedrin polypeptide and two minor 47 and 49 kDa polypeptides. A variety of diagnostic tools have been reported for this virus including real-time PCR and LAMP-based detection. Taxonomic position is still uncertain and International Committee on Taxonomy of Viruses lists MBV as a tentative species named PemoNPV in the genus Nucleopolyhedrovirus. However, according to the latest genomic information on the virus, it has been suggested to create a new group of non-occluded bacilliform viruses called nudiviruses with MBV as one of the members. The aim of the current work is to describe the knowledge on the status, distribution and host-range, pathology, transmission, virus structure and morphogenesis, genomic characteristics, diagnosis and the latest taxonomic position of MBV.
Penaeid shrimp; Viral disease; Monodon baculovirus; MBV
Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.
To avert the health problems induced by many environmental pollutants, available antioxidants have been evaluated. The present study was aimed to investigate whether α-tocopherol could protect the hexavalent chromium (Cr VI)-induced peroxidation in the liver and kidney and to explore the underlying mechanism of the same.
Materials and Methods:
A total of 24 Wistar adult female rats were equally divided into four groups. Group 1 served as control while Groups 2 and 3 were administered K2Cr2O7(10 mg/kg b.wt. s.c. single dose). In addition to (Cr VI), Group 3 also received α-tocopherol (125 mg/kg, daily) by oral gavage for 14 days. Group 4 was maintained as α-tocopherol control (dose as above). At the end of 14 days, blood samples were drawn for hematology. Subsequently, all the rats were sacrificed to collect liver and kidney samples for assay of tissue peroxidation markers, antioxidant markers and functional markers and histopathology.
Administration of chromium (Cr VI) in Group 2 significantly (P < 0.05) reduced the antioxidant markers such as superoxide dismutase and reduced glutathione along with significant (P < 0.05) increase in peroxidation markers such as malondialdehyde and protein carbonyls in the liver and kidney as compared with other groups. The functional markers in serum such as total protein was decreased significantly (P < 0.05), whereas other functional markers viz. alanine transaminase, blood urea nitrogen and creatinine were increased significantly (P < 0.05) in Group 2 as compared with the other groups. Significant (P < 0.05) decrease in hemoglobin, packed cell volume, total erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin and total leukocyte count were observed in Cr VI treated Group 2 rats. Prominent pathological changes were observed in the liver and kidney of Group 2. Co-treatment with α-tocopherol in Group 3 rats significantly (P < 0.05) reversed the Cr VI induced changes. The parameters in the study in Group 4 did not differ as compared with Group 1.
α–tocopherol exhibited protective effect against Cr VI-induced damage to the liver and kidney by inhibition of lipid peroxidation owing its antioxidant activity.
α-tocopherol; chromium (Cr VI); kidney; lipid peroxidation; liver
Lanthanides; Peptides; Fluorescent Probes; Protein Labeling; Microscopy
There is a need to improve treatments for metastatic breast cancer. Here we show activation of the phosphoinositide 3-kinase (PI3K) and MAP kinase (MAPK) pathways in a MMTV-CreBRCA1f/fp53+/− mouse model of breast cancer. When treated with the pan-Class IA PI3K-inhibitor NVP-BKM120, tumor doubling was delayed from 5 to 26 days. NVP-BKM120 reduced AKT phosphorylation, tumor cell proliferation and angiogenesis. Resistant tumors maintained suppression of AKT phosphorylation but exhibited activation of the MAPK-pathway at the “pushing margin”. Surprisingly, PI3K-inhibition increased indicators of DNA damage, poly-ADP-ribosylation and γH2AX, but decreased Rad51 focus formation, suggesting a critical role of PI3K activity for Rad51 recruitment. PARP-inhibitor Olaparib alone attenuated tumor growth modestly; however, the combination of NVP-BKM120 and Olaparib delayed tumor doubling to more than 70 days in the mouse model and over 50 days in xenotransplants from human BRCA1-related tumors, suggesting that combined PI3K- and PARP-inhibition might be effective treatment for BRCA1-related tumors.
PI3 Kinase inhibitor; NVP-BKM120; PARP-Inhibitor; Olaparib; BRCA1-related breast cancer
The World Health Organization (WHO) declared eradication of the dreadful disease “smallpox” in 1980. Though the disease has died down, the causative virus “variola” has not, as it has been well preserved in two high security laboratories—one in USA and another in Russia. The debate on whether the remaining stocks of the smallpox virus should be destroyed or not is ongoing, and the World Health Assembly (WHA) in 2011 has decided to postpone the review on this debate to the 67th WHA in 2014. A short questionnaire-based inquiry was organized during a one-day stem cell meeting to explore the views of various health care and life science specialists especially students on this aspect. Among the 200 participants of the meeting, only 66 had answered the questionnaire. 60.6% of participants who responded to the questionnaire were for preserving the virus for future reference, while 36.4% of the participants were for destroying the virus considering the magnitude with which it killed millions. However, 3% of the respondents were not able to decide on any verdict. Therefore, this inquiry expresses the view that “what we cannot create, we do not have the right to destroy.”
To identify correlates of anaemia during the first trimester of pregnancy among 366 urban South Indian pregnant women.
Cross-sectional study evaluating demographic, socio-economic, anthropometric and dietary intake data on haematological outcomes.
A government maternity health-care centre catering predominantly to the needs of pregnant women from the lower socio-economic strata of urban Bangalore.
Pregnant women (n 366) aged ≥18 and ≤40 years, who registered for antenatal screening at ≤14 weeks of gestation.
Mean age was 22.6 (SD 3.4) years, mean BMI was 20.4 (SD 3.3) kg/m2 and 236 (64.5%) of the pregnant women were primiparous. The prevalence of anaemia (Hb <11.0g/dl) was 30.3% and of microcytic anaemia (anaemia with mean corpuscular volume <80fl) 20.2%. Mean dietary intakes of energy, Ca, Fe and folate were well below the Indian RDA. In multivariable log-binomial regression analysis, anaemia was independently associated with high dietary intakes of Ca (relative risk; 95% CI: 1.79; 1.16, 2.76) and P (1.96; 1.31, 2.96) and high intake of meat, fish and poultry (1.94; 1.29, 2.91).
Low dietary intake of multiple micronutrients, but higher intakes of nutrients that inhibit Fe absorption such as Ca and P, may help explain high rates of maternal anaemia in India.
Anaemia; Pregnancy; Correlates; South India
Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.
Heart Failure; Perfusion; Microcirculation
Diarrhea; Campylobacter jejuni; antimicrobial resistance; India; bacteria
In high dose rate (HDR) brachytherapy, the source dwell times and dwell positions are vital parameters in achieving a desirable implant dose distribution. Inverse treatment planning requires an optimal choice of these parameters to achieve the desired target coverage with the lowest achievable dose to the organs at risk (OAR). This study was designed to evaluate the optimum source step size and maximum source dwell time for prostate brachytherapy implants using an Ir-192 source. In total, one hundred inverse treatment plans were generated for the four patients included in this study. Twenty-five treatment plans were created for each patient by varying the step size and maximum source dwell time during anatomy-based, inverse-planned optimization. Other relevant treatment planning parameters were kept constant, including the dose constraints and source dwell positions. Each plan was evaluated for target coverage, urethral and rectal dose sparing, treatment time, relative target dose homogeneity, and nonuniformity ratio. The plans with 0.5 cm step size were seen to have clinically acceptable tumor coverage, minimal normal structure doses, and minimum treatment time as compared with the other step sizes. The target coverage for this step size is 87% of the prescription dose, while the urethral and maximum rectal doses were 107.3 and 68.7%, respectively. No appreciable difference in plan quality was observed with variation in maximum source dwell time. The step size plays a significant role in plan optimization for prostate implants. Our study supports use of a 0.5 cm step size for prostate implants.
High dose rate brachytherapy; inverse optimization; adoptive volume optimization; step size; dwell time
We describe a draft genome sequence for Pediococcus acidilactici strain D3, a component of multistrain commercial cultures with biopreservative and biocontrol properties in food-based applications. Strain D3 encodes at least one antimicrobial peptide, pediocin AMPd3. The AMPd3-encoding operon exhibits high sequence similarity to the archetype pediocin, PA-1, encoded by P. acidilactici PAC 1.0.
Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive tumor type, different from smooth muscle and neural tumors of the gastrointestinal tract (GIT). They constitute the majority of gastrointestinal mesenchymal tumors of the GIT and are known to be refractory to conventional chemotherapy or radiation. They are defined and diagnosed by the expression of a proto-oncogene protein detected by immunohistochemistry which serves as a crucial diagnostic and therapeutic target. The identification of these mutations has resulted in a better understanding of their oncogenic mechanisms. The remarkable antitumor effects of the molecular inhibitor imatinib have necessitated accurate diagnosis of GIST and their distinction from other gastrointestinal mesenchymal tumors. Both traditional and minimally invasive surgery are used to remove these tumors with minimal morbidity and excellent perioperative outcomes. The revolutionary use of specific, molecularly-targeted therapies, such as imatinib mesylate, reduces the frequency of disease recurrence when used as an adjuvant following complete resection. Neoadjuvant treatment with these agents appears to stabilize disease in the majority of patients and may reduce the extent of surgical resection required for subsequent complete tumor removal. The important interplay between the molecular genetics of GIST and responses to targeted therapeutics serves as a model for the study of targeted therapies in other solid tumors. This review summarizes our current knowledge and recent advances regarding the histogenesis, pathology, molecular biology, the basis for the novel targeted cancer therapy and current evidence based management of these unique tumors.
Gastrointestinal stromal tumors; c-KIT; Imatinib mesylate; Surgery; Review
Primary open-angle glaucoma is associated with elevated intraocular pressure, which in turn is believed to result from impaired outflow of aqueous humour. Aqueous humour outflow passes mainly through the trabecular meshwork (TM) and then through pores formed in the endothelium of Schlemm's canal (SC), which experiences a basal-to-apical pressure gradient. This gradient dramatically deforms the SC endothelial cell and potentially contributes to the formation of those pores. However, mechanical properties of the SC cell are poorly defined. Using optical magnetic twisting cytometry and traction force microscopy, here we characterize the mechanical properties of primary cultures of the human SC cell, and for the first time, the scope of their changes in response to pharmacological agents that are known to modulate outflow resistance. Lysophosphatidic acid, sphingosine-1-phosphate (S1P) and thrombin caused an increase in cell stiffness by up to 200 per cent, whereas in most cell strains, exposure to latrunculin A, isoproterenol, dibutryl cyclic-AMP or Y-27632 caused a decrease in cell stiffness by up to 80 per cent, highlighting that SC cells possess a remarkably wide contractile scope. Drug responses were variable across donors. S1P, for example, caused 200 per cent stiffening in one donor strain but only 20 per cent stiffening in another. Isoproterenol caused dose-dependent softening in three donor strains but little or no response in two others, a finding mirrored by changes in traction forces and consistent with the level of expression of β2-adrenergic receptors. Despite donor variability, those drugs that typically increase outflow resistance systematically caused cell stiffness to increase, while in most cases, those drugs that typically decrease outflow resistance caused cell stiffness to decrease. These findings establish the endothelial cell of SC as a reactive but variable mechanical component of the aqueous humour outflow pathway. Although the mechanism and locus of increased outflow resistance remain unclear, these data suggest the SC endothelial cell to be a modulator of outflow resistance.
aqueous humour; cell mechanics; mechanobiology; outflow resistance; primary open-angle glaucoma; Schlemm's canal
The immunomodulatory activity of mangiferin was studied in various groups of animals. For this study, adult Swiss albino male mice were treated with benzo(a)pyrene, abbreviated as B(a)P, at 50 mg/kg body weight orally twice a week for 4 weeks; and mangiferin was also given orally (pre- and post-initiation of carcinoma) at 100 mg/kg body weight. Immunocompetence and immune complexes as measured by phagocyte index, avidity index, and soluble immune complex (SIC) levels (p<0.001), as well as NBT reduction, were decreased in the B(a)P-treated animals;whereas increased levels of immunocompetence were noted in the mangiferin-treated animals given B(a)P (p<0.001, p<0.05). The levels of immunoglobulins such as IgG and IgM were decreased considerably (p<0.001) in the B(a)P-treated animals compared with their levels in the control animals; whereas the IgA level was increased (p<0.001). In the mangiferin-treated experimental animals given B(a)P, the levels of IgG and IgM were significantly (p<0.001, p<0.05) increased whereas the IgA level was decreased compared with those for the B(a)P-treated mice. Oxidative changes in lymphocytes, neutrophils, and macrophages were also measured. The enhanced lipid peroxidation and decreased catalase and superoxide dismutase activities found in the lymphocytes, polymorphonuclear cells (PMN), and macrophages from B(a)P-treated mice were significantly reduced and increased, respectively, by the mangiferin treatment. This study confirms the immunomodulatory effect of mangiferin and shows an immunoprotective role arbitrated through a reduction in the reactive intermediate-induced oxidative stress in lymphocytes, neutrophils, and macrophages.
polyaromatic hydrocarbon; Benzo(a)Pyrene; mangiferin; immunomodulation
To evaluate and compare the variations in the inclination of occlusal plane of casts mounted on Artex articulator using a facebow with a fixed value and customized nasion indicator.
Materials and Methods:
Twenty two subjects were selected for this investigation. Two maxillary impressions were made, and casts poured. For each of the twenty two subjects, the facebow records were made with, Artex face-bow using a fixed value nasion indicator and customized nasion indicator and mounted. The angle between the occlusal plane of upper cast and the upper articulator arm was measured with a Universal bevel protractor and compared with the gold standard cephalometric value.
It shows that, when angle was measured between maxillary occlusal plane and upper member of the articulator, on the mounted cast using a customized nasion indicator and fixed value nasion indicator against the gold standard cephalometric value as a whole, it was found to be not significant. But, if each patient were evaluated individually, there found to be the difference in the angle.
Discussion and Conclusion:
Variation in occlusal plane was very minimal and close to the cephalometric value when using customized nasion indicator compared to fixed value nasion indicator on the Artex.
Articulator; cephalometry; facebow; nasion; occlusal plane
Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.
In both health and disease, human tissues are composed of a multiplicity of cellular types, many of which unknown and uncharacterized. Accurate, quantitative measurements of tissue cell composition are difficult to perform, as classic analytical techniques, such as flow cytometry and immunohistochemistry, depend on the availability of antigen-specific monoclonal antibodies and allow only for a few markers to be tested in parallel on each cell. As a result, the full repertoire of cell types contained in a tissue, and their relative abundance in physiological and pathological situations, remain largely unexplored. We developed a novel analytical platform for the study of tissue cell composition, based on single-cell gene-expression RT-PCR (SINCE-PCR). We tested the precision, accuracy and sensitivity of the method, and we used it to investigate the cell composition of human colon epithelia, both normal and cancerous. Starting from primary surgical specimens, we obtained single-cell suspensions of colon epithelial cells and we used flow cytometry to sort them one-by-one, in arrays of hundreds of single cells. We then exploited a microfluidic platform to analyze each individual cell for the expression of 96 genes in parallel, and we used statistical clustering algorithms to associate cells with similar gene-expression profiles. This novel approach led to the discovery of new epithelial cell populations and novel biomarkers to differentially label them. Moreover, it showed that, in human colon tumors, the heterogeneity observed among cancer cells closely mirrors the cellular diversity observed in normal epithelia, where immature progenitor cells intermingle with distinct subsets of mature goblet cells and enterocytes. Finally, SINCE-PCR analysis of monoclonal colon cancer xenografts, obtained from injection of a single (n = 1) “cancer stem cell” into immunodeficient mice, formally proved that this diversity is epigenetic in nature and originates from multi-lineage differentiation processes, reminiscent of physiological stem-cell systems.
A total of 874 fecal specimens (446 diarrheal cases and 428 controls) from diarrheal children admitted in the Infectious Diseases Hospital, Kolkata and age and sex matched asymptomatic subjects from an urban community were assessed for the prevalence of enterotoxigenic Bacteroides fragilis (ETBF). Isolates of B. fragilis were tested for the presence of enterotoxin gene (bft) by PCR. The detection rate of ETBF was 7.2% (63 of 874 specimens) that prevailed equally in diarrheal cases and controls (7.2% each; 32 of 446 cases and 31 of 428 controls). Male children up to one year age group was significantly (p<0.05) associated with ETBF infection as compared to children > 2 years of age in cases and controls. In 25 ETBF isolates, the bft gene was genotyped using PCR-RFLP and only two alleles were identified with prevalence rate of 40% and 60% for bft-1 and bft-3, respectively. All the ETBF isolates were susceptible for chloramphenicol and imipenem but resistant to clindamycin (48%), moxifloxacin (44%) and metronidazole (32%). Resistance of ETBF to moxifloxacin (44%) and metronidazole is an emerging trend. Pulsed-field gel electrophoresis (PFGE) revealed that majority of the ETBF isolates are genetically diverse. In the dendrogram analysis, two clusters were identified, one with ETBF resistant to 5–8 antimicrobials and the other cluster with metronidazole and moxifloxacin susceptible isolates from diarrheal cases. To our knowledge, this is the first detailed report on ETBF from India indicating its clinical importance and molecular characteristics.